Lectures 3-4: Reactive Oxygen Species I & II

Reactive Oxygen Species (ROS)

Oxygen containing chemically reactive molecules

Many have one unpaired electron (free radicals)

Free radicals

Molecules with one unpaired electron

Highly reactive and steals/binds another molecules electron

Short living

Radical ROS examples

*Oxygens all have one free electron

Superoxide: O₂^.-

Hydroxyl: ^.OH

Peroxyl: RO₂^.

Alkoxyl: RO^.

Hydroperoxyl: HO₂^.

Non-radical ROS examples

Hydrogen peroxide: H₂O₂

Hypochlorous acid: HOCl^-

Ozone: O₃

Singlet oxygen: ¹O₂

Peroxynitrite: ONOO^-

Strong oxidising potential

Main source of ROS

Oxidative phosphorylation - electron transport chain in mitochondria

0.1-2% of electrons passing through chain are incompletely reduced → superoxide radicals

Free radicals produced by OxPhos

H₂O₂

^.OH

O₂^.-

Superoxide dismutase (SOD) function

Detoxifies superoxides

O₂^.- → H₂O₂

Glutathione peroxidase (GPX) function

Converts hydrogen peroxide into water

H₂O₂ → H₂O

Non-enzymatic ROS production pathway

Non-enzymatic production via oxidative phosphorylation

Damage control via SOD & GPX

Enzymatic ROS production pathway

Enzymatic production via peroxisomes & phagosomes

Enzymes in peroxisomes generate ROS

Balance mediated by catalase

ROS generation in peroxisomes

Energy metabolism enzymes within the organelle

Fatty acid degradation via oxidation (H loss) leads to ROS production

H₂O₂ produced by oxidases (most common)

ie. Acyl-CoA oxidases O₂ → H₂O₂ via FAD → FADH₂

Catalase function

Converts H₂O₂ into H₂O

Maintains ROS balance

How ROS accumulation is avoided while keeping enough H₂O₂ needed for cellular signalling pathways

Respiratory Burst

Large productions of ROS used to neutralise pathogens

Executed by phagocytes & NADPH oxidase complex

NADPH oxidase complex

Found in cell membranes and membranes of phagosomes in neutrophils

Produces superoxide (O₂^.)

Superoxide, H₂O₂ & acids induce the destruction of pathogens

Neutrophil NADPH oxidase produces superoxide at a 90% higher rate than other cells

Exogenous ROS sources

Radiation - UV light, x-rays & γ-rays

Chemical reactions that form peroxides O₃ & O₂

Chemicals that promote superoxide formation ie. quinones, nitroaromatics & bipyridylium herbicides (paraquat)

Chemicals metabolised to radicals ie. polyhalogenated alkanes, phenols & aminophenols 

Xenobiotics (pesticides)

Chemicals that release iron ie. ferritin (Fenton reaction)

Redox signalling

Process where ROS & other reactive molecules act as chemical (second) messengers

Second messenger traits

Short lived so concentrations can change rapidly

Enzymatically generated in response to a stimulant

Enzymatically degraded or inactivated

Free radical second messengers

O₂^.-

H₂O₂

NO^. 

Platelet-derived growth factor (PDGF)

Induces cell proliferation

Downstream signalling induces superoxide & other ROS production which activate MAPK & TFs to then induce cell proliferation

Redox signalling pathways

NO retrograde messenger involved in neural plasticity & long term potentiation 

Superoxide regulates TFs in proliferation & inflammation

Tyr-phosphatase & membrane receptor (PDGF, insulin) regulation

JNK & MAPK signalling pathways

3 areas of ROS damage

Lipid

Protein

DNA

Lipid damage via ROS

Decreases number of double bonds in unsaturated FAs

Decreases membrane fluidity

Lipid peroxidation products can lead to cell damage

Protein damage via ROS

Caused by radicals accumulating or damage at a specific part of protein (transition metal ion binding to protein)

DNA damage via ROS

Oxidising radicals can attack DNA ie. radiation damage

Nuclear DNA damage leads to mutations & cannot replicate unless it avoids the repairs systems

ROS can cause DNA fragmentation, Y Chromosome microdeletions, epigenetic abnormalities, mtDNA damage & telomere attrition/shortening (seen in ageing)

mtDNA damage

Worse than nuclear DNA damage b/c it doesn’t have repair mechanisms or histones & produces the most ROS

Damage to mitochondrial function/integrity leads to further ROS release

Protein oxidation via ROS

Amino acid oxidation or post translational modification oxidation

OH added to sulphur in cysteine or threonine

Oxidative mods can change phys & chem properties ie. conformation, structure, solubility, susceptibility to proteolysis & enzyme activities

Further causes protein cross-linking, degradation & aggregation

Lipid peroxidation

Oxidative degradation of lipids

Free radical chain reaction mechanism

Targets polyunsaturated FAs ie. phospholipids

3 steps: initiation, propagation & termination

Initiation step of lipid peroxidation

Free radical steals an electron from a C-H bond in PUFA turning it into a fatty acid free radical (unstable)

R-O^. → R-OH

ie. OH^. forms H₂O

Propagation step of lipid peroxidation

Unstable fatty acid radical reacts w/ O₂ & forms peroxyl-fatty acid radical

Change in conformation occurs

O^. on peroxyl-fatty acid radical attacks H on another PUFA & creates a different fatty acid radical (unstable) & cycle continues

First PUFA becomes a fatty acid hydroperoxide 

Termination step of lipid peroxidation

Radical chain reaction mechanism stops when 2 radicals react & produce a non-radical species at high radical concentrations or by antioxidants Vit C & E donating a hydrogen

Whole membrane regions can be rapidly oxidised if termination does not occur

Ion channels & ROS damage

Alters ion channel activity

Increased intracellular Na²⁺ causes increased water → cellular swelling

Increased Ca²⁺ damage mitochondria & cause cellular hardening/arterial plaque or cell death

Voltage-gated ion channel dysfunction leads to neural malfunction

Malondialdehyde (MDA)

Product of arachidonic acid peroxidation

Mutagenic; can cause mutations to DNA in cell

Causes protein cross-linking & mitochondrial dysfunction

4-hydroxy-2-nonenal (HNE)

Product of linoleic acid peroxidation

Stable

Causes impaired protein degradation & mitochondrial dysfunction

Can move between cell & membrane & damages other sites

Forms covalent binding w/ proteins found in senile plaques

Associated w/ Alzheimer’s & Parkinson’s

Antioxidant compound

Substance that can compete w/ oxidisable substrates to significantly delay or inhibit their oxidation at low concentrations

Vitamin C & E function in lipid peroxidation termination

Tocopherol (Vit E) donates H from its hydroxyl group to reduce free radical

Inserted into cell membranes via its hydrophobic chain

Tocopherol radical is restored by ascorbate (Vit C)

They are less dangerous as free radicals than PUFAs and are neutralised later by other antioxidants ie. scavengers or enzymes

Scavenger antioxidants

Intercept chain reaction

Vitamin E (tocopherol) - fat soluble

Vitamin C (ascorbate) - water soluble

Glutathione 

Lipoic acid

Vitamin A (retinol) - fat soluble

Enzyme antioxidants

Prevents chain reaction

Superoxide dismutase (SOD)

Glutathione peroxidase (GPX)

Catalase

Hydroxyl radical

^.OH

Most damaging ROS

Produced by H₂O₂ reacting w/ Fe²⁺ in Fenton reaction

Attacks lipids, proteins & DNA causing tissue damage

Transferrin

Binds free iron (Fe²⁺)

Prevents Fenton reaction

Acts as a chelator & reduces risk of hydroxyl radical formation

Chelator

Chemical compound that binds metal ions tightly, forming a stable ring-like structure that traps the metal within the complex

SOD subtypes

FeSOD: predominantly in prokaryotes & chloroplasts

MnSOD: in mitochondria

Cu/ZnSOD: mostly in cytosol & peroxisomes

Ascorbate (Vitamin C) scavenging

Scavenges O₂^., ¹O₂ & H₂O₂ 

Chain breaking antioxidant 

Can be pro-oxidant at high concentrations & in the presence of metal ions

Water soluble so it functions best in aqueous phase of cell

Regenerates tocopherol from α-tocopheroxyl radical

Glutathione scavenging

Scavenges peroxides

Cellular reductant

Regenerates ascorbate radical

Regulator of gene expression

ROS damage repair

Polymerases, glycosylases & nucleases repair nuclear DNA

Proteases, peptidases & lipases repair proteins & lipids via reduction & degradation

Apoptosis & necrosis

PLA₂ can remove oxidised FAs

Cyt P450 isoforms may mediate the lysis of an oxidised PUFA → truncated FA left moves from inside membrane to extracellular compartment forming whiskers

Whiskers can be detected by receptors on macrophages for engulfment

Ageing definition

Progressive decline in physiological function, characterised by progressive decline of physiological function & increased susceptibility to disease and eventually death

Syndrome of changes that are deleterious, progressive, universal and currently irreversible

Ageing damage occurs to molecules (DNA, proteins, lipids), cells & organs

Theories of ageing

Two types: genetic theories & damage-accumulation theories

Rate of living theory (Pearl, 1928)

Animals die sooner if they live faster

Larger animals live longer & have lower resting metabolic rates

Small animals burn through their lifetime expenditure of energy per gram of tissue more quickly than larger animals and therefore die sooner

Harman’s theory in 1950s

ROS leads to cell damage & function deterioration

For longevity, mitochondrial rate of free radical production is more important than metabolic rate

10x less ROS in vitro

SOD discovery weakened scepticism

1950s theory evidence

Caloric restriction decreases ROS production & increases lifespan

In fruit flies: ageing is slowed by overexpression of catalase & SOD Methuselah strain are oxidative stress resistant & live 35% longer

Mice w/ IGF signalling defect are smaller, colder, have enhanced antioxidant defences & live longer
Losing IGF receptor increases oxidative stress resistance & longevity
SOD2^+/- mice have more oxidative damage & reduced lifespans

Mitochondrial dysfunction theory (Harman, 1972)

Mitochondria are the main producers AND targets of ROS

Decreased DNA repair & damaged & mutant mitochondrial proteins caused by ROS lead to further decline in cell function incl. apoptosis, cell death & ageing

mtDNA damage causes decreased energy production & more ROS production → ageing

Alzheimer’s & Parkinson’s & mtDNA deletions

Higher levels of mtDNA deletions found in cortical tissue of aged AD patients

Higher levels of mtDNA deletions found in the striatum of Parkinson’s patients

Aged rats w/ impaired long-term potentiation

Decreased PUFA & arachidonic acid levels do to ROS found in aged rats w/ impaired long-term potentiation

Protein oxidation & ageing

Protein oxidation increases w/ ageing

Increased carbonyl groups from direct arachidonic acid oxidation or ROS-mediated peptidergic cleavage found in hippocampus & cortex of aged animals

Increased 3-nitro-tyrosine found in cortex of aged animals

Caloric restriction

Consuming only 60-70% of normal food intake during early growth significantly increases lifespan as new physiological state is adopted

Smaller body size & delayed maturation, lower body temp, blood glucose, insulin levels, body fat & weight & increased daytime activity decreases ROS & age-related diseases

Caloric restriction & oxidative stress

Proposed caloric restriction:

Reduces oxidative stress as mitochondria produce less ROS while consuming the same amount of O₂

Prevents changes in gene expression that occur w/ age, promoting basal attenuation of heat-shock protein expression & increasing Hsp70 expression levels

Some studies claim it increases SOD, GPX & catalase activity in aged animals

Oxidative stress & CV disease

Ca²⁺ overload leads to atherosclerosis, vasoconstriction leading to hypertension, myocardial cell damage in ischemia & cardiac hypertrophy in heart failure

Atherosclerotic lesions

Atherosclerotic lesions can have increased Fe²⁺ & Ca²⁺ levels which further increases oxidative stress and ROS production (Fenton reaction)

Treated by reintroduction of blood & oxygen to the cells

Inflammatory dysregulation

Unbalanced inflammatory response, leads to chronic inflammation

Activated w/ harmless stimulus

Lack of inactivation

Targeting self tissues

Microglia & ROS

Microglia are the immune cells of the CNS

Microglia are largest source of ROS in the brain

Resting microglia → activated w/ infectious agents, neurotoxins & inflammatory mediators

IL-1, IL-6 & TNFα from activated microglia produce ROS

Chronic inflammation in the brain leads to ROS accumulation

Causes oxidative damage & neuronal death in neurological diseases

Traits consistent in motor neuron diseases

Increased ROS, lipid peroxidation, protein nitrosylation & iron deposits

Increased glia activation - H₂O₂, ^.NO & peroxynitrite

Alzheimer’s & ROS connections

SOD, catalase & GPX activity are lower in affected areas of the brain

ROS production is associated w/ amyloid plaques

ROS from increased microglial activity are implicated in beta-amyloid toxicity in cultured neurons

Parkinson’s disease

Characterised by loss of dopaminergic neurons in the substantia nigra responsible for the motor symptoms

Evidence of ROS involvement

Dopamine production & ROS production

Tyrosine → L-DOPA → Dopamine

Dopamine is synthesised via tyrosine hydroxylase & DOPA decarboxylase 

Tyrosine hydroxylase is Fe-dependent

Unstable reaction that undergoes oxidation & releases ROS (H₂O₂)

Hydrogen peroxide can travel to other neurons & interact w/ Fe (Fenton)

Proteins & lipids involved in the transport, release & uptake of dopamine are targets of ROS ie. VMAT2

Dopamine oxidation

Oxidised dopamine produces dopamine quinones that produce superoxide & aminochrome (leads to cell death)

Oxidised dopamine also produces neuromelanin that interacts w/ α-synuclein (increased levels found in Parkinson’s patients)

Mitochondrial dysfunction & Parkinson’s

Parkinson’s associated genes affect mitochondrial functions

Leads to increase in ROS formation & susceptibility

ie. Parkin & PINK1 are involved in the Ubiquitination-proteasome system so damage in those genes = impaired protein degradation in mitochondria